1. Field of the invention
The invention relates to and has among its objects the provision of antibodies which are specific for cytokinins having a glycosylated isoprenoid side chain and immunoassay methods for the determination of these cytokinins utilizing the antibodies.
2. Description of the art
Phytohormones regulate many aspects of plant growth and development. Plants, like all higher organisms, senesce, and there is evidence that a complex genetic program for senescence exists and is mediated by the interaction of naturally occurring phytohormones. An important class of phytohormones is the group of cytokinins having a purine ring, hereinafter denoted simply as cytokinins. These compounds are known to evoke a diversity of responses in plants. In addition to occurring in higher plants as free compounds, cytokinins also occur as component nucleotides in tRNA of plants, animals, and microorganisms, and in plant viral RNA (D. S. Letham and L. M. S. Palni, Annual Review of Plant Physiology 34: 163-197 (1983). The most important naturally occuring cytokinin is zeatin, shown in FIG. 1. It has a purine moiety (adenine), an isoprenoid side chain (6-(4-hydroxy-3-methylbut-trans-2-enyl), and a hydrogen attached to the 4' oxygen. Another important, naturally occurring cytokinin is dihydrozeatin. As shown in FIG. 2, it differs from zeatin in that the double bond of the isoprenoid side chain (6-(4-hydroxy-3-methylbutyl)) is saturated. Derivatives of zeatin and dihydrozeatin include compounds having a glycoside substituent (e.g., glucose, ribose) linked to the 4' oxygen of the isoprenoid side chain. These compounds are denoted as cytokinins having a glycosylated isoprenoid side chain. Illustrative compounds are shown in FIGS. 1 and 2. Zeatin and dihydrozeatin and their O-glycosylated derivatives can also have N-substitution on the purine ring. Examples include N-glycosides (compounds having a sugar moiety linked to the 7 or 9 nitrogen of the purine ring) or N-amino acyl forms (compounds having an amino acid substituent on the 7 or 9 nitrogen). The N-glycosides may be optionally phosphorylated to mono-, di-, or triphosphate esters.
Cytokinins having a glycosylated isoprenoid side chain differ from other cytokinins with regard to stereochemistry, metabolism, and function. The glycosylated side chain is substantially bigger than non-glycosylated side chain (FIG. 3), and the isoprenoid moiety of the side chain is partially masked by the O-glycoside. Thus, the antigenicity of these compounds (that is, their ability to react with receptors of the immune system to trigger antibody formation) differs from the antigenicity of the non-O-glycosides. With regard to differences in metabolism, hydrolysis of cytokinins having a glycosylated isoprenoid side chain is catalyzed by glycosidases which are well documented in the plant literature. In the absence of specific enzymes, these cytokinins can also be hydrolyzed under acidic conditions, especially at elevated temperatures. With regard to function, some studies suggest that cytokinins having a glycosylated isoprenoid side chain function as inactive, storage forms of cytokinins, which can be metabolically activated by enzymatic hydrolysis to the non-O-glycosylated forms.
While the significance of the multiple forms of cytokinins is not fully known, they do not have identical activities in various bioassays. For example, N-ribosides are active in most bioassays, and N-glucosides are inactive in these assays and appear to be dead-end metabolites. Because the different cytokinins do not have the same biological activity, it is important to measure all of the major forms present in a plant or other experimental system (Letham and Palni, 1983, supra). The effective concentration of even a single cytokinin is influenced by the physical sequestration of the molecule within the plant and transport processes which translocate it to different sites. Interconversions of cytokinins in the leaves, for example, could regulate the effective concentration of cytokinins in the chloroplast. The distinct chemical forms appear to have different functions, with ribosides the predominant translocatable form and glucosides the storage forms (Letham and Palni, 1983, supra). Ribotides are also found, sometimes in relatively high concentrations (I. M. Scott and R. Horgan, Planta 161: 345-354 (1984) and J. Badenoch-Jones et al., Plant Physiology 75: 1117-1125 (1984)).
There are several factors which cause difficulty in the mesurement of cytokinins. One problem is that cytokinins occur in low concentration in plant tissues (nanomolar concentration is typical). Another problem is that, as stated above, they exist in multiple chemical forms in the presence of many other molecules. Further, glycosides and nucleotide forms are inherently unstable to some methods of extraction and concentration. Physicochemical methods of quantitation of cytokinins involve elaborate purification procedures, and utilize complex and expensive instrumentation for detection. While quantitation by such methods is possible, it requires relatively large samples and is labor-intensive and time consuming. Other widely used methods are bioassays, which generally involve cell or organ cultures or growth of whole seedlings. One problem inherent in a bioassay is interference due to the presence of inhibitors of cytokinin activity. A serious problem of the bioassay procedures is that the activity of the multiple forms of cytokinins is not identical from assay to assay. Another problem is that typical bioassays are time consuming (several days) and are not adaptable to automation.
Polyclonal antibodies directed against cytokinins have been reported and have been used for immunoassay (E. W. Weiler, Annual Review of Plant Physiology 35: 85-95 (1984)), immunoaffinity fractionation (E. M. S. MacDonald and R. O. Morris, Methods in Enzymology 110: 347-358 (1985)), and immunocytochemical localization of cytokinins (M. E. Zavala and D. L. Brandon, Journal of Cell Biology 97: 1235-1239 (1983)). Monoclonal antibodies have also been reported which bind to cytokinins, with the first reports being E. J. Trione and R. O. Morris, Plant Physiology 72 (supplement): 114 (1983); and M. L. Woodsworth et al., Biochemical and Biophysical Research Communications 114: 791-796 (1983). No antibodies have been reported which bind to cytokinins having a glycosylated isoprenoid side chain. Therefore, the immunochemical methods using known antibodies do not permit measurement, fractionation, or localization of this important group of cytokinins. J. Badenoch-Jones et al., supra, first reported that antibodies specific for zeatin and zeatin riboside do not bind efficiently to cytokinins having a glycosylated isoprenoid side chain.
Cytokinins are low molecular weight compounds and are not themselves immunogenic. To obtain an immune response they must be coupled to an immunogenic carrier. Such compounds are known as haptens. In the studies reported by all investigators except D. L. Brandon et al., 1987, discussed infra, the antibodies to cytokinins were elicited using oxidized ribosides as haptens, that is, compounds with reactive aldehyde groups generated by the oxidative cleavage of the 9-N-ribosyl moiety of the corresponding cytokinin riboside. These compounds readily combine with amino groups on a protein carrier, and can then be reduced to form stable amines. These haptens cannot be used to make antibodies to cytokinins having a glycosylated isoprenoid side chain since such reactions would cleave the glycoside and eliminate this important feature of the molecule.
Brandon et al., in J. E. Fox and M. Jacobs (eds.) Molecular Biology of Plant Growth Control, 209-217, Alan R. Liss, Inc., New York (1987), used 9-(2-carboxyethyl) zeatin as hapten for eliciting antibodies to zeatin (a nonglycosylated cytokinin). This report does not disclose a hapten suitable for eliciting antibodies to cytokinins with a glycosylated isoprenoid side chain. None of the prior art teaches methods for obtaining such a compound. Cytokinins having a glycoslyated isoprenoid side chain do not have a functional group (other than the glycoside moiety) available for coupling to a carrier. Coupling through the glycoside moiety would destroy the key feature of the molecule in the process of making the immunogenic conjugate. It was unknown whether a suitable functional group could be introduced into a cytokinin having a glycosylated isoprenoid side chain to make a suitable derivative, or hapten, for conjugating to a carrier. The glycoside moiety is labile, and reactions such as hydrolysis, racemization, and oxidation would destroy the hapten.
Even if a functional group could be introduced, there were major uncertainties about the survival of the glycoside moiety during conjugation to carrier. Oxidative reactions or acidic conditions would be likely to hydrolyze the glycoside or cause racemization. Widely used carbodiimide reagents are not appropriate for coupling polyhydroxy compounds (such as cytokinins having a glycosylated isoprenoid side chain) because of side reactions, such as formation of O-alkyl isoureas (A. Williams and I. T. Ibrahim, Chemical Reviews 81: 589, 600 (1981) discusses this point as part of a comprehensive review of the reactions of carbodiimides.)
Even if conjugates could be prepared, the prior art does not disclose whether they would be sufficiently stable to reach receptors which trigger antibody production and whether the mouse (or any other species) would recognize it as an antigen and produce antibody. One problem is steric hindrance of the hapten which would alter or prevent antigenicity. Attachment to a protein would sterically hinder part of the hapten and could prevent its interaction with the cells and receptors of the immune system needed to trigger antibody production. The glycoside moiety itself partially masks the isoprenoid side chain (see FIG. 3), which is known to be recognized as an antigen. In view of the foregoing, it could not be predicted whether antibodies could be obtained having the ability to simultaneously recognize a purine ring, an isoprenoid side chain, and a 4'-O-glycoside.
As stated above, understanding how cytokinins regulate the physiological functions of plants necessitates quantitation of all of the forms of cytokinins. In addition, sites of synthesis and action, signals which regulate these processes, and transport phenomena must be characterized. Because of the importance of cytokinin interconversions, these problems must be addressed with analytical methods for cytokinins having a glycosylated isoprenoid side chain. Presently, immunoassays to quantitate these compounds are conducted by measuring zeatin, for example, before and after treatment with a glycosidase which hydrolyzes the glycosidic bond. Obtaining the measurement indirectly as the difference between two assay results is inherently less precise than performing a direct measurement. In addition, antibodies specific for cytokinins having a glycosylated isoprenoid side chain would provide ideal analytical tools to measure the enzymes which synthesize or degrade these cytokinins and growth regulatory substances which modulate the activity of these enzymes. Antibodies specific for cytokinins having a glycosylated isoprenoid side chain would provide the ideal reagents for these immunochemical methods.